sodium acetate precipitation times - (Jul/07/2006 )

Hi, I am using 100% ethanol and 3M sodium acetate to precipitate and purify cycle sequencing products for analysis on an automated cycle sequencer. I have been precipitating the reactions at -20degrees overnight, is this too long a precipitation? I have been told it may lead to loss of larger DNA products?
Any thoughts gratefully received, thanks

-phdstudentnewcastle-

I mix 100% ethanol and sodium acetate and leave it at at -80C for 5 min. It works nicely.

if in a hurry I just mix with ethanol & sodium acetate and if I c a white ppt., I centrifuge them immediately. Works only if there is lot of DNA.

-scolix-

QUOTE (scolix @ Jul 7 2006, 07:40 PM)

Thanks, is it bad to leave it for a long time though? I have been getting mixed sequencing results and am trying to rule this out as a reason.

-phdstudentnewcastle-

do you add a coprecipitant (glycogen)?

we allow the samples to precipitate, at -20C or lower, for 1 hour or longer. i'm not sure if anybody has allowed it to go overnight but as long as the tube is sealed and the ethanol doesn't evaporate, i don't see why the larger dna fragments would resolubilize.

-mdfenko-

I have precipitated over the weekend before with no problem at all, at both -20 and -80
(excessive time, I know that's not required, but I usually do 1/2 day or overnight if I think the amount of product is very low, to increase yield...not really necessary but it works for me)

if you have good yield and aren't worried about retention, I agree that you don't even really have to put it in the cold at all

-aimikins-

QUOTE (phdstudentnewcastle @ Jul 7 2006, 03:29 PM)

What sequencing chemistry do you use? I'm used to make sequencing reactions with Big Dye terminator (AppliedBiosystems) and purify them with ethanol/EDTA protocol suggested by Applied (see in the pdf version of Big Dye manual). This was the one that gave me the best results.

-Mari_P-

Thanks for all the comments. I use a Beckman Quickstart dye terminating kit, and purify with ethanol and sodium acetate only, no glycogen.

-phdstudentnewcastle-

QUOTE (phdstudentnewcastle @ Jul 7 2006, 02:55 PM)

Thanks for all the comments. I use a Beckman Quickstart dye terminating kit, and purify with ethanol and sodium acetate only, no glycogen.

we use the same kit and always use the glycogen (if for no other reason but to see the pellet, we are very visual here). it does improve the pelleting of the reaction products and will be more reproducible.

-mdfenko-